The alpha-subunit of the glycoprotein hormones in normally expressed in pituitary thyrotrophs and gonadotropes, and in placental cells. The goal of the proposed research project is to elucidate the mechanisms responsible for alpha-subunit gene expression in thyrotrophs. Our knowledge of the murine alpha-subunit gene structure, or preliminary determination of the location of the functionally important cis-acting elements in cell s of thyrotropic origin, and the availability of a pure murine alpha-subunit secreting thyrotropic ell line are crucial to attaining this goal. the investigator proposes to develop four specific aims. The first is to further localize the cis-acting elements between - 480 and -381 of the alpha-subunit gene promoter that determine thyrotroph-specific activation. The initial strategy will be to introduce mutations into the regions of the alpha-subunit gene promoter that have been shown to interact with thyrotroph nuclear proteins and determine the effect of these mutations on both functional and binding activity as examined in transient transfections and DNase I protection analysis, respectively. The second specific aim will optimize conditions for invito transcription complementation assays using extracts from the alpha-TSH cell line which enhance alpha-subunit promoter activity in non- thyrotropic cell extracts. In vitro transcription critical to our successful execution of this aim. The third specific aim will be to purify alpha-subunit transcription factors utilizing the alpha-TSH cell as a source of nuclear proteins containing those factor which are required for alpha-subunit activation. Our preliminary data suggest that the nuclear factors in alpha-TSH cells which interact with the alpha- subunit promoter are identical to those in native thyrotropic tumor cells. Our fourth specific aim will be to use the method of ligand screening to identify from cDNA expression libraries the thyrotroph- specific proteins which bind to the alpha-subunit promoter and activate is expression. The factors detected will be tested for their ability to bind to the cis-acting elements in a manner identical to that of thyrotropic tumors and alpha-TSH cell nuclear extracts. To establish the functional competence of newly identify factors, thyrotroph-dependent alpha subunit expression will be reconstituted in non-alpha-subunit expressing cell systems using transient transfection and in vitro transcription assays. Acquisition of new knowledge from this proposal will be important in elucidating mechanisms that contribute to alpha- subunit expression and thus TSH biosynthesis.